This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include loading then touching a pin or capillary to a surface, such as described in U.S. Pat. No. 5,807,522 or deposition by firing from a pulse jet such as an inkjet head, such as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere. Such a deposition method can be regarded as forming each feature by one cycle of attachment (that is, there is only one cycle at each feature during which the previously obtained biopolymer is attached to the substrate). For in situ fabrication methods, multiple different reagent droplets are deposited by pulse jet or other means at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array substrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides, and may also use pulse jets for depositing reagents. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence can be considered as multiple ones of the following attachment cycle at each feature to be formed: (a) coupling a selected nucleoside (a monomeric unit) through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (xe2x80x9cdeprotectionxe2x80x9d) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner. Conventionally, a single pulse jet or other deposition unit is assigned to deposit a single monomeric unit.
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in xe2x80x9cSynthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivativesxe2x80x9d, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach. The substrates are typically functionalized to bond to the first deposited monomer. Suitable techniques for functionalizing substrates with such linking moieties are described, for example, in Southern, E. M., Maskos, U. and Elder, J. K., Genomics, 13, 1007-1017, 1992.
In the case of array fabrication, different monomers may be deposited at different addresses on the substrate during any one cycle so that the different features of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each cycle, such as the conventional oxidation and washing steps in the case of in situ fabrication of polynucleotide arrays.
In array fabrication, the quantities of polynucleotide available are usually very small and expensive. Additionally, sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down as accurately as possible in the desired target pattern, are of the correct size, and that the DNA is uniformly coated within the feature. If any of these conditions are not met within a reasonable tolerance, the results obtained from a given array may be unreliable and misleading. This of course can have serious consequences to diagnostic, screening, gene expression analysis or other purposes for which the array is being used.
However, in any system used to fabricate arrays with the required small features, there is inevitably some degree of error, either fixed (and hence repeated) and/or random. In the case of both the deposition of previously obtained biopolymers, but particularly in the in situ fabrication method, drop deposition errors from cycle to cycle may be different and are cumulative in determining errors in the finally formed features. For example, polynucleotide arrays formed by the in situ method will have actual features represented only by the region where droplets of nucleoside monomers have overlapped (that is, the intersection of the nucleoside monomer droplets deposited during multiple cycles). The present invention realizes that in the conventional in situ system where a single pulse jet deposits all of a particular nucleoside monomer unit during a cycle, a serious trajectory error in just one such pulse jet will result in a serious error in the resulting feature (that is, the feature will be seriously smaller than expected). Furthermore, if one such pulse jet fails to fire during a single cycle at a feature, the resulting feature will effectively be useless (since it will be capped in the capping step or, where no capping step is used, will be missing a nucleotide and therefore will have the wrong sequence). It has been known to use multiple firings of a same reagent from a same pulse jet, during a same cycle. While this reduces random errors which might occur during a pulse jet firing, it does not correct for a fixed trajectory error of a pulse jet, nor will it correct for failure of that pulse jet.
It would be desirable then to provide a means by which serious errors in features formed during an in situ or any array fabrication method, can be reduced. It would further be desirable if the results of fixed errors in a drop deposition unit (such as a pulse jet of a multiple pulse jet system) can be reduced.
The present invention further realizes that serious errors in features during array formation, resulting from a serious error (including failure) of a drop deposition unit in a multiple drop deposition unit system, can be reduced by using multiple different drop deposition units to deposit the same reagent in any given cycle. As a result, while a feature in such a case may not be ideal, the error which might otherwise occur can be substantially reduced.
The present invention then, provides a method of forming an addressable array on a substrate. For each of multiple addresses on the substrate, a reagent drop set is deposited during a cycle so as to attach a corresponding moiety for that address. The foregoing step can be repeated as required, until the addressable array is formed. The reagent drop set deposited during one or more cycles for one or more of the multiple addresses includes, for a corresponding cycle and address, drops of a same reagent (which drops may or may not be of the same total composition) which are deposited from different deposition units during the same cycle. Such a method can be used where the array has features of any chemical moieties. For example, where the features are polymers, such as polynucleotides or peptides, the drops of the same reagent may include a same polymer (where the array is being formed in one cycle for each address by deposition of the previously obtained polymer, such as a polynucleotide or peptide) or a same monomeric unit (where the array is being formed in multiple cycles for each address during the in situ method, such as a same nucleoside monomer or same amino acid monomer).
In one aspect of the method, at least some of the deposition units are in a first line. The deposition units or the substrate, or both, is moved along a line parallel to the first line so as to form an addressable array with features in a line parallel to the first line. The drops of a same reagent deposited from different deposition units during the same cycle for an address, are deposited from different deposition units along the first line. The first line may, for example, be a straight or curved line.
In a particular configuration, the deposition units may be part of a same head so as to move as one unit, with at least the head being moved with respect to the substrate.
The present invention also provides an apparatus for forming an addressable array on a substrate, which can execute one or more methods of the present invention. In one aspect, the apparatus includes a deposition system having multiple deposition units (for example, pulse jets) each of which can dispense a reagent drop. A transport system moves at least one of the deposition system or the substrate. A processor controls the deposition system and the transport system. The processor control is such that for each of multiple addresses on the substrate, a reagent drop set is deposited by the deposition system during a cycle so as to attach a corresponding moiety for that address. Further, the processor control causes the foregoing step to be repeated if required, until the addressable array is formed. The processor control also ensures that the reagent drop set deposited during one or more cycles for one or more of the multiple addresses includes, for a corresponding cycle and address, drops of a same reagent which are deposited from different deposition units during the same cycle.
In the apparatus at least some of the deposition units may be in a first line and the apparatus includes a transport system to move at least one of the deposition system or substrate, both as described above. In this case, the processor may cause the drops of the same reagent to be deposited from different deposition units along the first line, during the same cycle for an address.
A further aspect of the present invention provides a method of determining a reagent drop deposition pattern for forming an addressable array. In this method, a target layout (sometimes referenced as an xe2x80x9caim layoutxe2x80x9d) for an addressable array is obtained. The reagent drop deposition pattern is determined from the target layout and the number of drop deposition units in a drop deposition system which includes multiple deposition units. By xe2x80x9cdeterminedxe2x80x9d in the foregoing aspect is referenced that the drop deposition pattern is obtained using the described items, but additional elements can also be taken into account. The determined drop deposition pattern includes a definition of, for each of multiple addresses on the substrate, a reagent drop set which is to be deposited during a cycle so as to attach a corresponding moiety for that address. The determined pattern further includes repetitions of the foregoing step as required, until the addressable array is formed. In the determined pattern, the reagent drop set deposited during one or more cycles for one or more of the multiple addresses includes, for a corresponding cycle and address, drops of a same reagent which are deposited from different deposition units during the same cycle.
The present invention further provides a computer program product which can execute any one or more methods of the present invention. Optionally, the present invention may further provide for exposing the array to a sample, and interrogating the array following the exposure and optionally processing results of the interrogation. Such an interrogation or processing result may be forwarded to a remote location. The present invention also provides a method in which data is transmitted representing a drop deposition pattern produced in one or more of the methods of the present invention.
The various aspects of the present invention can provide any one or more of the following and/or other useful benefits. For example, serious errors in features formed during an in situ or any array fabrication method, can be reduced. Further, the results of fixed errors in a drop deposition unit (such as a pulse jet of a multiple pulse jet system) can be reduced.